Method of increasing growth and yield in plants

ABSTRACT

Provided is a method of producing a genetically modified plant characterized as having increased growth and yield as compared to a corresponding wild-type plant comprising increasing the level of cyclin expression in the plant. The methods include providing a modified nucleic acid regulatory sequence from cycB1a;At resulting in increased gene transcription and expression. Also provided are modified nucleic acid regulatory sequences. Genetically modified plants characterized as having increased growth and yield are also provided.

This application is a continuation-in-part to U.S. application Ser. No.08/683,242, filed Jul. 18, 1996, the disclosure of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates generally to plant genetic engineering,and specifically to a method for producing genetically engineered plantscharacterized as having increased growth and yield.

BACKGROUND OF THE INVENTION

For each plant species, there exists a wide discrepancy in plant growthdue to environmental conditions. Under most conditions, the maximumgrowth potential of a plant is not realized. Plant breeding hasdemonstrated that a plant's resources can be redirected to individualorgans to enhance growth.

Genetic engineering of plants, which entails the isolation andmanipulation of genetic material, e.g., DNA or RNA, and the subsequentintroduction of that material into a plant or plant cells, has changedplant breeding and agriculture considerably over recent years. Increasedcrop food values, higher yields, feed value, reduced production costs,pest resistance, stress tolerance, drought resistance, the production ofpharmaceuticals, chemicals and biological molecules as well as otherbeneficial traits are all potentially achievable through geneticengineering techniques.

Plant growth responds to the increased availability of mineral nutrientsin the soil, but shoot and root growth respond differently. Moreover, adirect relationship between mineral nutrient availability and change ofgrowth rate is rarely observed over a larger concentration range. Thissuggest that plant growth is limited materially by nutrients requiredfor cell growth as well as by signaling pathways that control the rateof organ growth for the overall benefit of the plant. Although thecomponents of these regulatory pathways have not been identified, theydefine two distinct avenues to potentially improve plant growth. It hasbeen shown that enhanced accumulation of cyclin protein under control ofthe cdc2 promoter suffices to enhance root and overall plant growthunder non-limiting conditions on growth media.

Plants rarely grow under optimal conditions. Plant growth can be limitedby water availability, mineral nutrients and a short growing season.Drought tolerance in genetic variants of a given species is wellcorrelated with the penetration depth of its root system into the soil.Fertilizers are often not optimally utilized because of insufficientlypenetrating root systems. Although the induction of flowering can now becontrolled, thereby extending the potential growth range of someimportant crop species, this does not in itself lead to increasedbiomass.

The ability to manipulate gene expression provides a means of producingnew characteristics in transformed plants. For example, the ability toincrease the size of a plant's root system would permit increasednutrient assimilation from the soil. Moreover, the ability to increaseleaf growth would increase the capacity of a plant to assimilate solarenergy. Obviously, the ability to control the growth of an entire plant,or specific target organs thereof would be very desirable.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that increased growthand yield in plants can be achieved by elevating the level of cyclinexpression.

In a first embodiment, the invention provides a method of producing agenetically modified plant characterized as having increased growth andyield as compared to the corresponding wild-type plant. The methodincludes contacting a plant cell with a nucleic acid sequence comprisinga regulatory sequence, wherein said regulatory sequence is operablyassociated with a nucleic acid encoding a cyclin protein, to obtain atransformed plant cell; producing plants from said transformed plantcell; and selecting a plant exhibiting said increased yield. Inparticular, the nucleic acid can be a cyclin gene and the regulatorysequence can be a cyclin gene promoter, such as cycB1a;At.

In another embodiment, the invention provides a transformed plant cellor a transformed plant having a nucleic acid sequence having aregulatory sequence of cycB1a;At operably linked to a heterologousnucleic acid sequence.

In yet another embodiment, the invention provides an isolated nucleicacid sequence having a functional cycB1a;At regulatory sequence. In aparticular aspect, the regulatory sequence is the sequence set forth inSEQ ID NO:1.

The invention also provides plants, plant tissue and seeds produced bythe methods of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows steady state levels of cdc2aAt mRNA and p34 protein, panela; cyc1aAt mRNA during IAA induction of lateral root meristems, panel b;cyc1aAt mRNA in selected non-induced transgenic lines, panel c;normalized transcript levels relative to wild-type are indicated. Col-0,wild-type; 1A2, 2A5, 4A3, 11A1: T2 homozygous; 6A, 7A, 8A: T1heterozygous transgenic lines.

FIG. 2 shows an in situ hybridization analysis of cdc2aAt and cyc1aAttranscripts in root apices and developing lateral roots. Panels a-d showcross sections of quiescent roots (panels a,b) or proliferating cells inprimordia (panels c,d) that were hybridized to cdc2aAt (a) or cyc1aAt(b-d) anti-sense probes. Panels e, f show cyc1aAt mRNA abundance incontiguous meristematic cell files in root apices. Transcriptaccumulation is indicated by silver grain deposition and visualized byindirect red illumination. Scale bar is 10 μm in a-d, 5 μm in e. fc,founder cell accumulating cyc1aAt transcripts; p, pericycle cell layer;r, towards the root apex; s, towards the shoot.

FIG. 3 shows increased root growth rate in Arabidopsis thaliana (A.thaliana) ectopically expressing cyc1aAt cyclin. Panel a, Wild-type(left) or transgenic line 6A (T1 generation) containing thecdc2aAt::cyc1At gene fusion (right). Arabidopsis seed were plated on MS(3% sucrose) agar and grown in a vertical orientation for 7 d. Plantstransformed with the vector alone or with unrelated promoter::uidAconstructs or with a cdc2aAt::cyc1aAt fusion in which the cdc2aAt 5'untranslated leader was interrupted by a DS transposon insertion did notshow this phenotype. Panel b, wild-type (left) or transgenic line 6A (T1generation) (right) 6 d after IAA induction of lateral roots. Oneweek-old seedlings grown hydroponically were treated with 10 μMIAA_(eff) to stimulate lateral root development.

FIG. 4 shows aphidicolin (APC) release of the -1148 transformant and the-205 transformant.

FIG. 5 shows a series of constructs operably linked to cyc1a and GUSwherein a series of nested deletions were generated at the 5' end of thecycB1a;At regulatory sequence.

FIG. 6 shows a Northern blot analysis that shows elevation of endogenouslevels of cyclins following treatment of roots with thegrowth-stimulatory hormone auxin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods for increasing the yield of aplant, such as a agricultural crop, by elevating the cyclin expressionlevel in the plant. Increased cyclin expression in plant cells competentto divide results in increased plant growth.

In a preferred embodiment, the invention provides a method for producinga genetically modified plant characterized as having increased yield ascompared to a plant which has not been genetically modified (e.g., awild-type plant). The method comprises contacting plant cells withnucleic acid encoding a cyclin protein, wherein the nucleic acid isoperably associated with a regulatory sequence to obtain transformedplant cells; producing plants from the transformed plant cells; andthereafter selecting a plant exhibiting increased growth and yield.

In a further embodiment, the regulatory sequence of the invention isderived from a cycB1a;At gene. The regulatory sequence of the cycB1a;Atgene is approximately 1.2 kb in length. However, functional fragments ofthis regulatory sequence are provided which confer a modifiedtranscriptional activity upon nucleic acid sequence which are operablylinked to the regulatory sequence. By "modified transcriptionalactivity" is meant transcription of linked sequences above or belowwild-type expression of the linked sequence.

As used herein, the term "yield" or "plant yield" refers to increasedcrop growth, and/or increased biomass. In one embodiment, increasedyield results from increased growth rate and increased root size. Inanother embodiment, increased yield is derived from shoot growth. Instill another embodiment, increased yield is derived from fruit growth.

As used herein, the term "agronomic" includes, but is not limited to,changes in plant yield, growth or root size. Other agronomic propertiesinclude insect resistance, protein production, drought tolerance, andother factors desirable to agricultural production and business.

The term "genetic modification" as used herein refers to theintroduction of one or more exogenous nucleic acid sequences, e.g.,cyclin encoding sequences, as well as regulatory sequences, into one ormore plant cells, which can generate whole, sexually competent, viableplants. The term "genetically modified" as used herein refers to a plantwhich has been generated through the aforementioned process. Geneticallymodified plants of the invention are capable of self-pollinating orcross-pollinating with other plants of the same species so that theforeign gene, carried in the germ line, can be inserted into or bredinto agriculturally useful plant varieties. The term "plant cell" asused herein refers to protoplasts, gamete producing cells, and cellswhich regenerate into whole plants.

As used herein, the term "plant" refers to either a whole plant, a plantpart, a plant cell, or a group of plant cells, such as plant tissue orplant seed. Plantlets are also included within the meaning of "plant".Plants included in the invention are any plants amenable totransformation techniques, including gymnosperms and angiosperms, bothmonocotyledons and dicotyledons.

Examples of monocotyledonous angiosperms include, but are not limitedto, asparagus, field and sweet corn, barley, wheat, rice, sorghum,onion, pearl millet, rye and oats and other cereal grains. Examples ofdicotyledonous angiosperms include, but are not limited to tomato,tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce,peas, alfalfa, clover, cole crops or Brassica oleracea (e.g., cabbage,broccoli, cauliflower, brussel sprouts), radish, carrot, beets,eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers andvarious ornamentals. Examples of woody species include poplar, pine,sequoia, cedar, oak, etc.

The term "exogenous nucleic acid sequence" as used herein refers to anucleic acid foreign to the recipient plant host or, native to the hostif the native nucleic acid is substantially modified from its originalform. For example, the term includes a nucleic acid originating in thehost species, where such sequence is operably linked to a promoter thatdiffers from the natural or wild-type promoter. In the broad method ofthe invention, at least one nucleic acid sequence encoding cyclin isoperably linked with a promoter. It may be desirable to introduce morethan one copy of cyclin polynucleotide into a plant for enhanced cyclinexpression. For example, multiple copies of a cyclin polynucleotidewould have the effect of increasing production of cyclin even further inthe plant.

The term "regulatory sequence" as used herein refers to a nucleic acidsequence capable of controlling the transcription of an operablyassociated gene. Therefore, placing a gene under the regulatory controlof a promoter or a regulatory element means positioning the gene suchthat the expression of the gene is controlled by the regulatorysequence(s). In general, promoters are found positioned 5' (upstream) ofthe genes that they control. Thus, in the construction of promoter genecombinations, the promoter is preferably positioned upstream of the geneand at a distance from the transcription start site that approximatesthe distance between the promoter and the gene it controls in thenatural setting. As is known in the art, some variation in this distancecan be tolerated without loss of promoter function. Similarly, thepreferred positioning of a regulatory element, such as an enhancer, withrespect to a heterologous gene placed under its control reflects itsnatural position relative to the structural gene it naturally regulates.

Cyclin-encoding nucleic acids utilized in the present invention includenucleic acids encoding mitotic cyclins such as, for example, cyclin B;nucleic acids encoding S-phase cyclins such as, for example cyclin A,and nucleic acids encoding G1 phase cyclins. Specific cyclins which canbe utilized herein include cyc1aAt, cyc3aAt, cyc3aAt, cycB1a; At, cycd1,cycd2 and the like. Preferably, the nucleic acid used in the method ofthe invention encodes the cyc1aAt protein (Genebank Accession No.X62279).

Genetically modified plants of the present invention are produced bycontacting a plant cell with a nucleic acid sequence encoding thedesired cyclin. To be effective once introduced into plant cells, thecyclin-encoding nucleic acid must be operably associated with a promoterwhich is effective in plant cells to cause transcription of the cyclintransgene. Additionally, a polyadenylation sequence or transcriptioncontrol sequence, also recognized in plant cells, may also be employed.It is preferred that the nucleic acid be introduced via a vector andthat the vector harboring the nucleic acid sequence also contain one ormore selectable marker genes so that the transformed cells can beselected from non-transformed cells in culture, as described herein.

The term "operably associated" or "operably linked" refers to functionallinkage between a regulatory sequence, preferably a promoter sequence,and the cyclin-encoding nucleic acid sequence regulated by the promoter.The operably linked promoter controls the expression of the cyclinnucleic acid sequence.

The expression of cyclin genes employed in the present invention may bedriven by a number of promoters. Although the endogenous, or nativepromoter of a structural gene of interest may be utilized fortranscriptional regulation of the gene, preferably, the promoter is aforeign regulatory sequence.

Such regulatory sequences include the cycB1a;At regulatory sequence andfragments thereof. Such fragments include sequences about -1148 basesupstream of the transcriptional start site (i.e., -1), as well assequence from -1 to -60, -1 to -120, -1 to -205, -1 to -286, and -1 to-351. Modified regulatory sequences thus provides a method to deliverincreased levels of a desired gene of interest by modifying, forexample, repressor functions and enhancer functions or the regulatorysequence. For example, deletions of the cycB1a;At regulatory sequencesuch that repressor functions are removed can provide a regulatorysequences which results in increased gene expression. Such deletions ofthe cycB1a;At sequence include, but are not limited to, cyclin promotersdeleted to -286 or -205.

When it is desired to increase growth and yield in the whole plant,cyclin expression should be directed to all cells in the plant which arecapable of dividing. This can be accomplished by using a promoter activein all meristems. Such promoters include, for example, the cdc2apromoter and the cyc07 promoter. (See for example, Ito et al., PlantMol. Biol., 24:863, 1994; Martinez et al., Proc. Natl. Acad. Sci. USA,89:7360, 1992; Medford et al., Plant Cell, 3:359, 1991; Terada et al.,Plant Journal, 3:241, 1993; Wissenbach et al., Plant Journal, 4:411,1993).

When it is desired to increase growth and yield in a specific organ,cyclin expression should be targeted to the appropriate meristem, e.g.,the shoot meristem, the floral meristem, the root meristem etc. This canbe accomplished by using a tissue specific promoter. Examples of tissuespecific promoters active in shoot meristems are described in Atanassovaet al., Plant Journal, 2:291, 1992 and Medford et al., Plant Cell,3:359, 1991. Examples of tissue specific promoters active in floralmeristems are the promoters of the agamous and apetala 1 genes aredescribed in Bowman et at, Plant Cell, 3:749, 1991; and Mandel et al.,Nature, 360:273, 1992.

The particular promoter selected should be capable of causing sufficientcyclin expression to cause increased yield and/or increased biomass. Itshould be understood that cyclin expression can be altered in cells thatare competent to divide. The promoters used in the vector constructs ofthe present invention may be modified, if desired, to affect theircontrol characteristics. For example, deletions from the 5' end of thecycB1a;At regulatory sequence increases transcriptional activity.

Optionally, a selectable marker may be associated with thecyclin-encoding nucleic acid. As used herein, the term "marker" refersto a gene encoding a trait or a phenotype which permits the selectionof, or the screening for, a plant or plant cell containing the marker.Preferably, the marker gene is an antibiotic resistance gene whereby theappropriate antibiotic can be used to select for transformed cells fromamong cells that are not transformed. Examples of suitable selectablemarkers include adenosine deaminase, dihydrofolate reductase,hygromycin-B-phosphotransferase, thymidine kinase, xanthine-guaninephospho-ribosyltransferase and amino-glycoside 3'-O-phosphotransferaseII. Other suitable markers will be known to those of skill in the art.

To commence a transformation process in accordance with the presentinvention, it is first necessary to construct a suitable vector andproperly introduce it into the plant cell. Vector(s) employed in thepresent invention for transformation of a plant cell include acyclin-encoding nucleic acid sequence operably associated with apromoter, such as cycB1a;At or fragments thereof. Details of theconstruction of vectors utilized herein are known to those skilled inthe art of plant genetic engineering.

For plant expression vectors, suitable viral promoters include the 35SRNA and 19S RNA promoters of CaMV (Brisson, et al., Nature, 310:511,1984; Odell, et al., Nature, 313:810, 1985); the full-length transcriptpromoter from Figwort Mosaic Virus (FMV) (Gowda, et al., J. CellBiochem., 13D: 301, 1989) and the coat protein promoter to TMV(Takamatsu, et al., EMBO J. 6:307, 1987). Alternatively, plant promoterssuch as the light-inducible promoter from the small subunit of ribulosebis-phosphate carboxylase (ssRUBISCO) (Coruzzi, et al., EMBO J., 3:1671,1984; Broglie, et al., Science, 224:838, 1984); mannopine synthasepromoter (Velten, et al., EMBO J., 3:2723, 1984) nopaline synthase (NOS)and octopine synthase (OCS) promoters (carried on tumor-inducingplasmids of Agrobacterium tumefaciens) or heat shock promoters, e.g.,soybean hsp17.5-E or hsp17.3-B (Gurley, et al., Mol. Cell. Biol, 6:559,1986; Severin, et al., Plant Mol. Biol., 15:827, 1990) may be used.

Promoters useful in the invention include both natural constitutive andinducible promoters as well as engineered promoters. The CaMV promotersare examples of constitutive promoters. To be most useful, an induciblepromoter should 1) provide low expression in the absence of the inducer;2) provide high expression in the presence of the inducer; 3) use aninduction scheme that does not interfere with the normal physiology ofthe plant; and 4) have no effect on the expression of other genes.Examples of inducible promoters useful in plants include those inducedby chemical means, such as the yeast metallothionein promoter which isactivated by copper ions (Mett, et al., Proc. Natl. Acad. Sci., U.S.A.,90:4567, 1993); In2-1 and In2-2 regulator sequences which are activatedby substituted benzenesulfonamides, e.g., herbicide safeners (Hershey,et al., Plant Mol. Biol., 17:679, 1991); and the GRE regulatorysequences which are induced by glucocorticoids (Schena, et al., Proc.Natl. Acad. Sci., U.S.A., 88:10421, 1991). Other promoters, bothconstitutive and inducible will be known to those of skill in the art.

The particular promoter selected should be capable of causing sufficientexpression to result in the production of an effective amount ofstructural gene product, e.g., CDR1 polypeptide to cause increasedresistance to plant pathogens. The promoters used in the vectorconstructs of the present invention may be modified, if desired, toaffect their control characteristics.

Tissue specific promoters may also be utilized in the present invention.An example of a tissue specific promoter is the promoter active in shootmeristems (Atanassova, et al., Plant J., 2:291, 1992). Other tissuespecific promoters useful in transgenic plants, including the cdc2apromoter and cyc07 promoter, will be known to those of skill in the art.(See for example, Ito, et al., Plant Mol. Biol., 24:863, 1994; Martinez,et al., Proc. Natl. Acad. Sci. USA, 89:7360,1992; Medford, et al., PlantCell, 3:359, 1991; Terada, et al., Plant Journal, 3:241, 1993;Wissenbach, et al., Plant Journal, 4:411, 1993).

There are promoters known which limit expression to particular plantparts or in response to particular stimuli. For example, potato tuberspecific promoters, such as the patatin promoters or the promoters forthe large or small subunits of ADPglucose pyrophosphorylase, could beoperably associated with CDR1 to provide expression primarily in thetuber and thus, provide resistance to attacks on the tuber, such as byErwinia. A fruit specific promoter would be desirable to impartresistance to Botrytis in strawberries or grapes. A root specificpromoter would be desirable to obtain expression of CDR1 in wheat orbarley roots to provide resistance to Ggt. One skilled in the art willknow of many such plant part-specific promoters which would be useful inthe present invention.

Alternatively, the promoters utilized may be selected to confer specificexpression of CDR1 in response to fungal infection. The infection ofplants by fungal pathogens activate defense-related orpathogenesis-related (PR) genes which encode (1) enzymes involved inphenylpropanoid metabolism such as phenylalanine ammonia lyase, chalconesynthase, 4-coumarate coA ligase and coumaric acid 4-hydroxylase, (2)proteins that modify plant cell walls such as hydroxyproline-richglycoproteins, glycine-rich proteins, and peroxidases, (3) enzymes, suchas chitinases and glucanases, that degrade the fungal cell wall, (4)thaumatin-like proteins, or (5) proteins of as yet unknown function. Thedefense-related or PR genes have been isolated and characterized from anumber of plant species. The promoters of these genes may be used toobtain expression of CDR1 in transgenic plants when such plants arechallenged with a pathogen, particularly a fungal pathogen such as Pi.The particular promoter selected should be capable of causing sufficientexpression of CDR1 to result in the production of an effective amount ofpolypeptide.

Promoters used in the nucleic acid constructs of the present inventionmay be modified, if desired, to affect their control characteristics.For example, the CaMV 35S promoter may be ligated to the portion of thessRUBISCO gene that represses the expression of ssRUBISCO in the absenceof light, to create a promoter which is active in leaves but not inroots. The resulting chimeric promoter may be used as described herein.For purposes of this description, the phrase "CAMV 35S" promoter thusincludes variations of CaMV 35S promoter, e g., promoters derived bymeans of ligation with operator regions, random or controlledmutagenesis, etc. Furthermore, the promoters may be altered to containmultiple "enhancer sequences" to assist in elevating gene expression.

Cyclin-encoding nucleic acid sequences utilized in the present inventioncan be introduced into plant cells using Ti plasmids of Agrobacteriumtumefaciens (A. tumefaciens), root-inducing (Ri) plasmids ofAgrobacterium rhizogenes (A. rhizogenes), and plant virus vectors. (Forreviews of such techniques see, for example, Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, NY, SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9, and Horsch et al., Science, 227:1229,1985, both incorporated herein by reference). In addition to planttransformation vectors derived from the Ti or Ri plasmids ofAgrobacterium, alternative methods may involve, for example, the use ofliposomes, electroporation, chemicals that increase free DNA uptake,transformation using viruses or pollen and the use of microprojection.

One of skill in the art will be able to select an appropriate vector forintroducing the cyclin-encoding nucleic acid sequence in a relativelyintact state. Thus, any vector which will produce a plant carrying theintroduced cyclin-encoding nucleic acid should be sufficient. Even useof a naked piece of DNA would be expected to confer the properties ofthis invention, though at low efficiency. The selection of the vector,or whether to use a vector, is typically guided by the method oftransformation selected.

The transformation of plants in accordance with the invention may becarried out in essentially any of the various ways known to thoseskilled in the art of plant molecular biology. (See, for example,Methods of Enzymology, Vol. 153, 1987, Wu and Grossman, Eds., AcademicPress, incorporated herein by reference). As used herein, the term"transformation" means alteration of the genotype of a host plant by theintroduction of cyclin-nucleic acid sequence.

For example, a cyclin-encoding nucleic acid can be introduced into aplant cell utilizing A. tumefaciens containing the Ti plasmid, asmentioned briefly above. In using an A. tumefaciens culture as atransformation vehicle, it is most advantageous to use a non-oncogenicstrain of Agrobacterium as the vector carrier so that normalnon-oncogenic differentiation of the transformed tissues is possible. Itis also preferred that the Agrobacterium harbor a binary Ti plasmidsystem. Such a binary system comprises 1) a first Ti plasmid having avirulence region essential for the introduction of transfer DNA (T-DNA)into plants, and 2) a chimeric plasmid. The chimeric plasmid contains atleast one border region of the T-DNA region of a wild-type Ti plasmidflanking the nucleic acid to be transferred. Binary Ti plasmid systemshave been shown effective in the transformation of plant cells (DeFramond, Biotechnology, 1:262, 1983; Hoekema et al., Nature, 303:179,1983). Such a binary system is preferred because it does not requireintegration into the Ti plasmid of A. tumefaciens, which is an oldermethodology.

Methods involving the use of Agrobacterium in transformation accordingto the present invention include, but are not limited to: 1)co-cultivation of Agrobacterium with cultured isolated protoplasts; 2)transformation of plant cells or tissues with Agrobacterium; or 3)transformation of seeds, apices or meristems with Agrobacterium.

In addition, gene transfer can be accomplished by in plantatransformation by Agrobacterium, as described by Bechtold et al., (C.R.Acad. Sci. Paris, 316:1194, 1993) and exemplified in the Examplesherein. This approach is based on the vacuum infiltration of asuspension of Agrobacterium cells.

The preferred method of introducing a cyclin-encoding nucleic acid intoplant cells is to infect such plant cells, an explant, a meristem or aseed, with transformed A. tumefaciens as described above. Underappropriate conditions known in the art, the transformed plant cells aregrown to form shoots, roots, and develop further into plants.

Alternatively, cyclin-encoding nucleic acid can be introduced into aplant cell using mechanical or chemical means. For example, the nucleicacid can be mechanically transferred into the plant cell bymicroinjection using a micropipette. Alternatively, the nucleic acid maybe transferred into the plant cell by using polyethylene glycol whichforms a precipitation complex with genetic material that is taken up bythe cell.

Cyclin-encoding nucleic acid can also be introduced into plant cells byelectroporation (Fromm et al., Proc. Natl. Acad. Sci., U.S.A., 82:5824,1985, which is incorporated herein by reference). In this technique,plant protoplasts are electroporated in the presence of vectors ornucleic acids containing the relevant nucleic acid sequences. Electricalimpulses of high field strength reversibly permeabilize membranesallowing the introduction of nucleic acids. Electroporated plantprotoplasts reform the cell wall, divide and form a plant callus.Selection of the transformed plant cells with the transformed gene canbe accomplished using phenotypic markers as described herein.

Another method for introducing a cyclin-encoding nucleic acid into aplant cell is high velocity ballistic penetration by small particleswith the nucleic acid to be introduced contained either within thematrix of such particles, or on the surface thereof (Klein et al.,Nature 327:70, 1987). Bombardment transformation methods are alsodescribed in Sanford et al. (Techniques 3:3-16, 1991) and Klein et al.(Bio/Techniques 10:286, 1992). Although, typically only a singleintroduction of a new nucleic acid sequence is required, this methodparticularly provides for multiple introductions.

Cauliflower mosaic virus (CaMV) may also be used as a vector forintroducing nucleic acid into plant cells (U.S. Pat. No. 4,407,956).CaMV viral DNA genome is inserted into a parent bacterial plasmidcreating a recombinant DNA molecule which can be propagated in bacteria.After cloning, the recombinant plasmid again may be cloned and furthermodified by introduction of the desired nucleic acid sequence. Themodified viral portion of the recombinant plasmid is then excised fromthe parent bacterial plasmid, and used to inoculate the plant cells orplants.

As used herein, the term "contacting" refers to any means of introducinga cyclin-encoding nucleic acid into a plant cell, including chemical andphysical means as described above. Preferably, contacting refers tointroducing the nucleic acid or vector containing the nucleic acid intoplant cells (including an explant, a meristem or a seed), via A.tumefaciens transformed with the cyclin-encoding nucleic acid asdescribed above.

Normally, a plant cell is regenerated to obtain a whole plant from thetransformation process. The immediate product of the transformation isreferred to as a "transgenote". The term "growing" or "regeneration" asused herein means growing a whole plant from a plant cell, a group ofplant cells, a plant part (including seeds), or a plant piece (e.g.,from a protoplast, callus, or tissue part).

Regeneration from protoplasts varies from species to species of plants,but generally a suspension of protoplasts is first made. In certainspecies, embryo formation can then be induced from the protoplastsuspension. The culture media will generally contain various amino acidsand hormones, necessary for growth and regeneration. Examples ofhormones utilized include auxins and cytokinins. Efficient regenerationwill depend on the medium, on the genotype, and on the history of theculture. If these variables are controlled, regeneration isreproducible.

Regeneration also occurs from plant callus, explants, organs or parts.Transformation can be performed in the context of organ or plant partregeneration. (see Methods in Enzymology, Vol. 118 and Klee et al.,Annual Review of Plant Physiology, 38:467, 1987). Utilizing the leafdisk-transformation-regeneration method of Horsch et al., Science,227:1229, 1985, disks are cultured on selective media, followed by shootformation in about 2-4 weeks. Shoots that develop are excised from calliand transplanted to appropriate root-inducing selective medium. Rootedplantlets are transplanted to soil as soon as possible after rootsappear. The plantlets can be repotted as required, until reachingmaturity.

In vegetatively propagated crops, the mature transgenic plants arepropagated by utilizing cuttings or tissue culture techniques to producemultiple identical plants. Selection of desirable transgenotes is madeand new varieties are obtained and propagated vegetatively forcommercial use.

In seed propagated crops, mature transgenic plants can be self crossedto produce a homozygous inbred plant. The resulting inbred plantproduces seed containing the newly introduced foreign gene(s). Theseseeds can be grown to produce plants that would produce the selectedphenotype, e.g. increased yield.

Parts obtained from regenerated plant, such as flowers, seeds, leaves,branches, roots, fruit, and the like are included in the invention,provided that these parts comprise plant cells that have beentransformed as described. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

Plants exhibiting increased growth and/or yield as compared withwild-type plants can be selected by visual observation. The inventionincludes plants produced by the method of the invention, as well asplant tissue and seeds.

In another embodiment, the invention provides a method of producing aplant characterized as having increased growth and yield by contacting aplant capable of increased yield with a cyclin promoter-inducing amountof an agent which induces cyclin gene expression. Induction of cyclingene expression results in production of a plant having increased yieldas compared to a plant not contacted with the agent.

A "plant capable of increased yield" refers to a plant that can beinduced to express its endogenous cyclin gene to achieve increasedyield. The term "promoter inducing amount" refers to that amount of anagent necessary to elevate cyclin gene expression above cyclinexpression in a plant cell not contacted with the agent, by stimulatingthe endogenous cyclin promoter. For example, a transcription factor or achemical agent may be used to elevate gene expression from native cyclinpromoter, thus inducing the promoter and cyclin gene expression.

The invention also provides a method of providing increasedtranscription of a nucleic acid sequence in a selected tissue. Themethod comprises growing a plant having integrated in its genome anucleic acid construct comprising, an exogeneous gene encoding a cyclinprotein, said gene operably associated with a tissue specific wherebytranscription of said gene is increased in said selected tissue.

Plant development is plastic with post-embryonic organogenesis mediatedby meristems (Steeves and Sussex, Patterns in Plant Development, 1-388(Press Syndicate of the University of Cambridge, Cambridge, 1989)).Although cell division is intrinsic to meristem initiation, maintenanceand proliferative growth, the role of the cell cycle in regulatinggrowth and development is unclear. To address this question, theexpression of cdc2 and cyclin genes, which encode the catalytic andregulatory subunits, respectively, of cyclin-dependent protein kinasescontrolling cell cycle progression (Murray and Hunt, The Cell Cycle (NewYork), 1993) were examined. Unlike cdc2, which is expressed not only inapical meristems but also in quiescent meristems, (Martinez et al.,Proc. Natl. Acad. Sci. USA, 89:7360, 1992), transcripts of cyc1aAtaccumulated specifically in active meristems and dividing cellsimmediately before cytokinesis. Ectopic expression of cyc1aAt undercontrol of the cdc2aAt promoter in Arabidopsis plants markedlyaccelerated growth without altering the pattern of development orinducing neoplasia. Thus, cyclin expression is a limiting factor forgrowth.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLE 1

A full length cyc1aAt cyclin cDNA was placed under control of theArabidopsis cdc2aAt promoter (Hemerly et al., 1992, supra). The chimericgene was cloned into a T-DNA transformation vector carrying theselection marker hygromycin phospho-transferase (Hyg^(r)) andtransformed into Arabidopsis using the vacuum-infiltration method(Bechtold and Pelletier, Acad. Sci. Paris, Life Sci., 316:1194, 1993) tointroduce Agrobacterium tumefaciens. Several independent transgeniclines having elevated steady-state levels of cyc1aAt mRNA showed adramatic increase of both main and lateral root growth rate, correlatedwith proportionally increased fresh weight, dry mass and DNA content,but not cell size. Enhanced growth was orderly, with no observeddifferences in morphology and clearly not neoplastic.

Arabidopsis seedlings (ecotype Columbia) were grown in 20 ml MS medium(Murashige and Skoog, Physiol. Plant., 15:473, 1962). Eight- to10-day-old plants were transferred to MS medium buffered with 50 mMpotassium phosphate, pH 5.5, and initiation of lateral roots wasstimulated by addition of IAA to 10 μM_(eff) (non-dissociated IAA).Roots were collected at the time points indicated and total RNA andprotein isolated. 500 ng poly(A)+RNA was separated on 1% formaldehydegels (Ausubel et al., Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley-Interscience, New York, 1987),transferred to Nytran membranes (Schleicher and Schull) and hybridizedto ³² P-labeled probes corresponding to nucleotides (nt) 674-1004 ofcyc1aAt (Hemerly et al.,Proc. Natl. Acad. Sci. USA, 89:3295, 1992), ornt 661-1386 of Arabidopsis cdc2aAt (Hirayama et al., Gene, 105:159,1991), followed by hybridization with nt 2576-2824 of Arabidopsis UBQ3(Norris et al., Plant Mol. Biol., 21:895, 1993) for normalization. Blotswere quantified with a Molecular Dynamics Phosphorimager. cyc1aAt is asingle copy gene in Arabidopsis. Total protein was separated on 12%SDS-PAGE and transferred to PVDF membranes. p34^(cdc2aAt) was detectedwith serum raised in rabbits against the peptide YFKDLGGMP (SEQ IDNO:1), corresponding to amino acids 286-294, and visualized by EnhancedChemiluminescence Assay (Amersham).

FIG. 1 shows steady state levels of cdc2aAt mRNA and p34 protein, panela; cyc1aAt mRNA during IAA induction of lateral root meristems, panel b;cyc1aAt mRNA in selected non-induced transgenic lines, panel c;normalized transcript levels relative to wild-type are indicated. Col-0,wild-type; 1A2, 2A5, 4A3, 11A1: T2 homozygous; 6A, 7A, 8A: T1heterozygous transgenic lines. cyc1aAt mRNA levels in the lines 4A3, 6A,7A, 8A, and 3A exceeded those of IAA induced wild-type roots.

The levels of cdc2 mRNA and p34^(cdc2) protein per cell did not markedlychange following stimulation of lateral root initiation by the auxinindoleacetic acid (IAA) (FIG. 1, panel a). Hence, while cdc2 expressionis correlated with the competence to divide, root growth and initiationof lateral roots do not appear to be limited by the abundance of thecyclin-dependent protein kinase p34 catalytic subunit and, moreover,ectopic expression of cdc2 in transgenic Arabidopsis failed to perturbgrowth or development (Hemerly et al., EMBO J., 14:3925, 1995).

In contrast, IAA treatment of Arabidopsis roots induced the expressionof several cyc genes from low basal levels and in particular cyc1aAtmRNA, which encodes a mitotic cyclin (Hemerly et al., supra)), exhibiteda rapid 15 to 20-fold increase (FIG. 1, panel b).

FIG. 2 shows an in situ hybridization analysis of cdc2aAt and cyc1aAttranscripts in root apices and developing lateral roots. Panels a-d showcross sections of quiescent roots (panels a,b) or proliferating cells inprimordia (panels c,a) that were hybridized to cdc2aAt (a) or cyc1aAt(b-d) anti-sense probes. Panels e,f show cyc1aAt mRNA abundance incontiguous meristematic cell files in root apices. Transcriptaccumulation is indicated by silver grain deposition and visualized byindirect red illumination. Scale bar is 10 μm in a-d, 5 μm in e. fc,founder cell accumulating cyc1aAt transcripts; p, pericycle cell layer;r, towards the root apex; s, towards the shoot.

Tissue samples were processed for in situ hybridization to examineexpression of cyclin transcripts. The samples were treated with 10 μMIAA. After 8 or 24 h incubation, radish (Raphanus sativa var ScarletGlobe) roots were processed as described (Drews et al., Cell, 65:991,1991). Sections (8 μm) were hybridized to a ³³ P-labeled RNA probe,corresponding to nt 674-1004 of cyc1aAt (Hemerly et al., supra) (FIG. 2,panels b-e) or to a ³⁵ S-labeled probe used in a corresponding to nt661-1386 of cdc2aAt (Hirayama et al., supra), for 14 h at 48° C. in 50%formamide. After hybridization, the final washes were for 1 h at 58° C.in 0.015 m NaCl and slides were then exposed for 3 weeks (cyc1aAt) or 5d (cdc2aAt). After developing, silver grains were illuminated laterallywith red light, specimens were visualized by phase contrast and doubleexposures were taken on FUJI Velvia film. Images were assembled in ADOBEPhotoshop. For the analysis summarized in FIG. 2, panel f, silver grainswere counted and cell size measured in the cell file shown in FIG. 2,panel e.

In situ hybridization showed that, unlike cdc2, cyc1aAt transcripts werenot detected in quiescent pericycle cells, but accumulated in single,cytoplasmically dense cells of incipient lateral root primordia, and inthe emergent organ cyc1aAt was expressed exclusively in the meristem(FIG. 2, panels a-d). Moreover, crucifer roots consisted of long cellfiles that arise by transverse divisions followed by longitudinalexpansion (Dolan et al., Development, 119:71, 1993), and within such acontiguous spatial display of sequential cell division phases, cyc1aAttranscripts accumulated only in large cells immediately prior tocytokinesis, declining to background levels in the adjacent smalldaughter cells (FIG. 2, panels e,f). A similar, stringentspatio-temporal relationship of cyclin expression and mitosis wasobserved in Antirrhinum shoot apical meristems (Fobert et al., EMBO J.,13:616, 1994).

The close correlation between cyc1aAt expression and cell divisionduring growth of the root apical meristem and the initiation of lateralroots, together with the pattern of cyc1aAt promoter activity deducedfrom the expression of cyc1aAt::uidA gene fusions in transgenicArabidopsis (Ferreira et al., Plant Cell, 6:1763, 1994), suggested thatcyclin abundance might be a key factor regulating root growth anddevelopment. To test this hypothesis transgenic Arabidopsis weregenerated (Bechtold and Pelletier, Acad. Sci. Paris, Life Sci.,316:1194, 1993) containing cyc1aAt under control of the cdc2aAtpromoter. Five transformants were obtained in which the level of cyc1aAtmRNA in untreated roots exceeded that observed in IAA-stimulated rootsof wild-type plants (FIG. 1, panel c), and these lines were chosen forfurther study.

An NheI site was introduced in the third codon of the cyc1aAt cDNA by invitro mutagenesis and this open reading frame subsequently ligated tothe cdc2aAt promoter with an in vitro generated XbaI site at codon 3.This fragment was ligated into pBiB-Hyg (Becker et al., Pl. Mol. Biol.,20:1195, 1992) and transfected into Agrobacterium tumefaciens GV3101(Koncz and Schell, Mol. Gen. Genet., 204:383, 1986). Arabidopsisthaliana (A. thaliana) (ecotype Columbia) was transformed by vacuuminfiltration (Bechtold et al., supra), and transgenic seedlings (T0generation) were selected on MS plates containing 30 μg/ml hygromycin.52 independent transgenic lines were obtained and elevated levels ofcyc1aAt mRNA were detected in 9 of the 11 lines analyzed in detail.Growth assays were performed on heterozygous T1 and homozygous T2progeny as indicated.

FIG. 3 shows increased root growth rate in Arabidopsis ectopicallyexpressing cyc1aAt cyclin. Panel a, Wild-type (left) or transgenic line6A (T1 generation) containing the cdc2aAt::cyc1aAt gene fusion (right).Arabidopsis seed were plated on MS (3% sucrose) agar and grown in avertical orientation for 7 d. Plants transformed with the vector aloneor with unrelated promoter::uidA constructs or with a cdc2aAt::cyc1aAtfusion in which the cdc2aAt 5' untranslated leader was interrupted by aDS transposon insertion did not show this phenotype. Panel b, wild-type(left) or transgenic line 6A (T1 generation) (right) 6 d after IAAinduction of lateral roots. One week-old seedlings grown hydroponicallywere treated with 10 μM IAA_(eff) to stimulate lateral root development.

Strong expression of the cdc2aAt::cyc1aAt transgene caused a markedincrease in the rate of organized root growth (FIG. 3, panel a).Homozygous or heterozygous seed were plated on MS agar and plants grownin vertical orientation for 7 days with a 16 h day/8 h night schedule at22° C. Four images of each plate were acquired with a SpeedlightPlatinum frame grabber (Lighttools Research) at 24 h intervals and rootgrowth analyzed with NIH-Image by measuring the displacement of rootapices. Following growth analysis, roots from 10 plants of each classwere collected and RNA analyzed. To measure cell sizes, roots werecleared by overnight incubation in saturated chloral hydrate, visualizedwith Normarski optics, photographed and analyzed with NIH-Image.Statistical analysis (t-test with unpaired variances) was performed withMS Excel. Root growth in IAA-treated plants was assessed 3 and 6 d afterinduction by determination of fresh weight of roots excised fromliquid-grown plants and then dry weight following lyophilization for 24h. Total DNA was extracted from dried material (Ausubel et al., supra).

In heterozygous T2 progeny, increased growth rate, measured bydisplacement of the apex of the main root in time-lapse photography,strictly co-segregated with transgene expression and individuals lackingthe transgene grew at the same rate as wild-type plants (Table 1). Theaverage size of epidermal, cortical, endodermal and pericycle cells wasequivalent or slightly reduced in cdc2aAt::cyc1aAt transformantscompared to wild-type plants (Table 2), and hence increased growthreflects increased cell number rather than cell size. The pattern ofspontaneous lateral root initiation and overall root morphology wereindistinguishable in wild-type and transgenic plants (FIG. 3, panel a).When treated with 1 μM IAA, which induces well-separated lateral rootprimordia, the frequency of primordia initiated per unit length of themain roots was not altered (mean of 1.08 initials/mm with a standarddeviation of 0.09 in wild-type compared with 1.14+/-0.07 and 1.09+/-0.13in the two transgenic lines examined). However, growth and developmentof lateral roots following induction by 10 μM IAA, was markedlyaccelerated in the cdc2aAt::cyc1aAt transformants, giving rise to a muchenlarged root system (FIG. 3, panel b). Enhanced root growth incdc2aAt:cyc1aAt plants following IAA treatment superficially resemblesthe alf1 phenotype (Celenza et al., Genes & Development, 9:2131, 1995)and these plants have elevated levels of cyc1aAt transcripts but incontrast to cdc2aAt::cyc1aAt transformants, alf1 plants initiatesupernumerary lateral roots. The several-fold greater gain of freshweight in IAA-treated cdc2aAt::cyc1aAt plants compared to equivalentwild-type controls was accompanied by marked increased in DNA contentand dry weight (Table 3). Confocal microscopy confirmed that theenhanced growth response to IAA, which was also observed in severallines showing weaker cdc2aAt::cyc1aAt expression, did not reflecttransgene stimulation of cell vacuolation or elongation. Thus, ectopiccyclin expression enhances root growth by stimulation of cell divisionactivity in meristems, thereby increasing the rate of cell productionwithout altering meristem organization.

The data above indicate that cdc2aAt::cyc1aAt expression is sufficientto enhance growth from established apical meristems, suggesting thatcell cycle activity regulates meristem activity. However, the failure toinduce gratuitous organ primordia by ectopic expression of cyc1aAt undercontrol of the cdc2aAt promoter implies additional control points in thegeneration of a new apical meristem, either through post-translationalregulation of cyclin-dependent protein kinase activity or the operationof parallel regulatory pathways. In most animal cells, the commitment tocell division occurs late in G1 (Pardee, A. B., Science, 246:603, 1989),and cyclin D1 and cyclin E are rate-limiting for G1 progression incultured cells (Ohtsubo and Roberts, Science, 259:1908, 1993; Quelle etal., Genes Dev., 7:1559, 1993; Resnitzky and Reed, Mol. Cell Biol.,15:3463, 1995). Elevated levels of cyclin D1 are observed in severaltumors (Motokura et al., Nature, 350:512, 1991; Rosenberg et al., Proc.Natl. Acad. Sci. USA, 88:9638, 1991; Withers et al., Mol. Cell Biol.,11:4864, 1991) and ectopic expression in transgenic mice promoteshyperplasia and adenocarcinomas (Wang et al., Nature, 369:669, 1994).

In contrast, ectopic expression of cyc1aAt did not result in neoplasiabut stimulated organized growth, without altering meristem organizationor size as monitored by confocal microscopy. Moreover, morphology of thetransgenic plants was not altered and increased growth was accompaniedby accelerated organ development. Thus, cyclin expression is a crucial,limiting upstream factor in an intrinsic regulatory hierarchy governingmeristem activity, organized growth and indeterminate plant development.This regulatory hierarchy, which is distinctly different from that inanimals, where determinate development limits proliferative growth,exemplified by the strict morphogenetic control of cell division duringmuscle differentiation (Halevy et al., Science, 267:1018, 1995; Skapeket al., Science, 267:1022, 1995), may underlie the striking plasticityof plant growth and development (Drew, M. C., New Phytol., 75:479,1975). Cyclin abundance may function as a rheostat to allow flexiblegrowth control in response to changes in the environment such asnutrient availability.

                  TABLE 1                                                         ______________________________________                                                Root apical growth                                                              Rate           % of                                                   Plant line [μm · h.sup.-1 ] wild-type n                         ______________________________________                                        Col-0   (-)   254.1          100    57                                          2A5 (-) 253.1 99.6 56                                                         3A (+)  341.4* 134.4 20                                                       3A (-) 259.4 102.1 30                                                         4A3 (+)  291.6* 114.8 47                                                      6A (+)  354.1* 139.5 20                                                       6A (-) 252.4 99.3 16                                                          7A (+)  344.9* 135.7 24                                                       7A (-) 249.8 98.3 19                                                          8A (+)  335.4* 131.9 21                                                       8A (-) 258.6 101.8 31                                                         11A1 (-) 258.8 101.8 45                                                       2A5 (-) 253.1 99.6 56                                                       ______________________________________                                         Table 1 shows a comparison of root apical growth rates.                       Plant line = independent transformants (except for Col0).                     (+) = plants that show enhanced growth phenotype due to presence of           adequate levels of cyclinencoding nucleic acid.                               (-) = plants that have lost introduced cyclinencoding nucleic acid or do      not exhibit sufficient cyclin expression for enhanced growth.                 Rate = rate of displacement of root apex per unit time. (*denotes values      significantly different from WildType growth rate.)                           n = number of individual plants analyzed.                                

                  TABLE 2                                                         ______________________________________                                                        Plant line                                                                          7A           8A                                           Col-0  (transgenic)  (transgenic)                                                    (wild type)      Size       Size                                       Cell Type Size [μm] n [μm] n [μm] n                                ______________________________________                                        Epidermis                                                                              137       37     129   34   158   12                                   Cortex 159 31  135*  7 160  9                                                 Endodermis 109 23  90* 22 107 11                                              Pericycle  73 26  67 19  71 57                                              ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Growth of seedling root system                                                             Fresh weight  Dry weight  DNA per                                  Plant [mg]  [mg]  root [g]                                                  line     3d    6d        3d  6d      3d   6d                                  ______________________________________                                        Col-0    11     25       1.7  2.4    5    14                                    4A3 31 136 4.5 15.4 8 35                                                      6A 30 155 4.2 19.3 10  46                                                     7A 24 156 3.5 16.8 9 38                                                       8A 18 134 2.5 12.8 8 33                                                     ______________________________________                                         Table 2 shows a comparison of cell size; and Table 3 shows a comparison o     root growth after IAA treatment in wildtype and in transgenic Arabidopsis     lines containing the cdc2aAt::cyclaAt gene fusion. The lines 3A, 6A, 7A,      8A are heterozygous T1 populations with more than one introduced              transgene; (+) denotes plants with increased cyclaAt transcript levels,       (-) plants with wildtype cyclaAt transcript levels.                           The following T2 lines are homozygous for cdc2aAt:cyclaAt: 2A5, 4A3 and       11A1; constitutive cyclaAt expression in 4A3, but not in 2A5 and 11A1,        exceeds IAAinduced wildtype levels (FIG. 1). n, number of plants analyzed     *, means that are significantly different from the wildtype; for a, P <       0.001, for b, P < 0.01. Fresh weight = weight of freshly excised root         system. Dry weight = weight after 24 hours of drying.                    

One aspect of cyclin overexpression in the CDC2a::cyc1aAt transgenicplants is its role in stimulating cyclin-dependent kinase (CDK) complexactivity. Cyclins function to activate the catalytic subunit of suchcomplexes. Several lines of genetic evidence indicate that specificcyclin genes largely differ in their regulation during the cell cycle,but not in their biochemical ability to activate kinase activity. Thisevidence indicates that structurally divergent cyclins can functionallysubstitute for each other. First, the budding yeast genome encodes foronly one catalytic subunit of the cyclin-dependent kinase gene family(CDC28) involved in governing cell cycle progress. However, it encodesfor at least 9 cyclins which are only distantly related by structure butwhich nonetheless all activate CDK activity after associating with thep34cdc28 protein encoded by CDC28. Second, Drosophila or human cyclingenes have been isolated by way of complementation of a triple yeast G1cyclin (CLN) knockout strain. Not only were animal G1 cyclins isolatedbut mitotic cyclins were isolated as well. Recent research has alsoshown that yeast CLB or mitotic type cyclins can bypass the requirementfor CLN cyclins.

Taken together, these observations demonstrate that distinctlyregulated, and structurally only very distantly related cyclins (some ofthese share only 19% homology) substitute for one another such that theyare able to activate cyclin-dependent kinase activity by associationwith the catalytic subunit, albeit with varying degrees of efficiency.Thus, plant cyclins in general have the inherent property of enhancingroot and plant growth by virtue of their capacity to associate withendogenous proteins encoded by CDK homologous genes, and therefore thepresent invention applies to other plant cyclins beyond cyc1aAt.

FIG. 6 shows a Northern blot analysis that shows elevation of endogenouslevels of cyclins following treatment of roots with thegrowth-stimulatory hormone auxin. The blot shows that 5 differentmitotic cyclin genes and one G1 cyclin gene were all expressed atelevated levels upon auxin (IAA) treatment which in due course leads tothe initiation of a new root. The induction of many endogenous cyclingenes following growth stimulation leads to the conclusion that theregulatory pathways controlling growth in plants enhance cyclinexpression levels in general. Thus, cell division enhancement in thecourse of growth stimulation is not likely a unique property ofexpressing a specific cyclin gene or enhancing the level of its proteinproduct.

The emerging picture that cyclins exist in even larger gene families inplants than in yeast or animals is not unexpected (Renaudin et al. PlantMol. Biol. [1996] 32 1003-1018). Regulatory genes in plants tend toexist in large, distinctly regulated gene families. For example, theregulatory genes controlling pigment (anthocyanin) synthesis constitutegene families. Most of the bright red and blue colors found in higherplants are anthocyanins. Using corn as an example for the rest of theplant world, crucial regulatory genes of the helix-loop-helix class (R,B, Lc) and of the myb class (C1, P1) are required to stimulatetranscription of the biosynthetic genes of the anthocyanin pathway.These regulatory genes are functionally redundant, in other words theycan replace each other. However in maize, they are regulateddifferentially, such that P1 is required for anthocyanin pigmentation inthe vegetative tissue of the plant, and is sensitive to light intensity,whereas C1 operates in reproductive tissues and operates constitutively.Similar properties hold for the R, B, Lc family. Constitutive expressionof the maize R-gene suffices to induce ubiquitous anthocyaninbiosynthetic genes and pigment accumulation in the heterologous plantArabidopsis (Lloyd A M et al. Science, [1992] 258 1773-1775).

Another example for complex regulatory gene families in plants are themembers of the calmodulin-like domain protein kinase gene family (Hrabaket al. Plant Mol. Biol. [1996] 31 405-412). At least 12 such genes arenow known in Arabidopsis. These enzymes are all activated by calcium,but their expression patterns in the plant are quite distinct.

Taken together, plants have evolved complex multi-gene families ofregulatory genes that are functionally, i.e. biochemically, redundant,but differentially regulated. Thus, structurally divergent members ofgene families combine a common biochemical activity with differentialregulation likely to enable the plant to appropriately respond toenvironmental and developmental cues.

EXAMPLE 2

A 2.8 kb HindIII fragment including approximately 1.2 kb of upstreamsequence and genomic sequence corresponding to the first 306 amino acidsencoded by the cycB1a;At gene was isolated. 3' deletions of thisfragment were generated by exonuclease III treatment and a 1.8 kbfragment that included promoter sequence and coding sequence up to aminoacid 116 was selected to generate a translational fusion toβ-glucuronidase (GUS). Subsequently, a series of nested 5' deletions wasgenerated. The plasmids described here terminated at 351, 286, 205, 120,60 and 9 bp, respectively, upstream of the transcription start site. Theresulting series of constructs (FIG. 5) were cloned into the KpnI-SacIsites of the pBIB transformation vector (Becker et al., (1990), Nucl.Acids Res. 18:203) and subsequently introduced into tobacco BY2 cellsand Arabidopsis plants by Agrobacterium-medicated transformation (An,G., (1985), Plant physiol., 568-570; Bechtold et al., (1993), C.R. Acad.Sci. Paris, Life Sci. 316:1194-1199).

To establish a synchronized cell population, N. Tabacum BY2 (pCDG) cellswere arrested in S phase with the DNA polymerase inhibitor Aphidicolin(APC) (Kodama and Komamine, (1995), Meth. Cell Biol. 49:315-329).Following release from cell cycle arrest, samples were withdrawn everyhour for RNA analysis. In cells transformed with the -1148 or -351cyclin promoter-GUS construct, RNA abundance increased approximately50-fold as cells entered M-phase. However, in cells transformed withcyclin promoters deleted to -286 or -205, RNA abundance increasedapproximately 200-fold as cells entered M-phase (FIG. 4). Furtherremoval of 5' sequences substantially reduced the amplitude of cyclininduction (-120, -60 deletions), until cell cycle-regulated expressionwas lost after deletion to -9. Therefore, the DNA sequence between -351and -286 comprises a binding motif for a cognate protein with repressorfunction, the sequence between -205 and -60 comprises an activator motifand sequences between -60 and -9 are responsible for cell cycleregulation. Utilization of a cyclin promoter deleted to -286 or -205therefore provides a method to deliver increased levels of a desiredgene of interest specifically at G2/M.

The XhoI site present within the third cyclin exon was use to exchangethe E. coli UidA coding sequence with the remainder of the cyclin cDNA.A series of constructs with 5'-deleted promoters was used to transformArabidopsis and evaluate the growth properties of the resultant plants.Nine out of 13 independent transgenic lines that have elevated steadystate levels of cycB1a;At mRNA show a 10-25% increase of root growthrate, as well as increased fresh weight and dry mass but no cell size.When grown on soil, overall plant growth, specifically the rate of leafgrowth is dramatically enhanced, although neither final size of theplant nor flowering time are strongly affected. Thus, increased growthearly during leaf development results in a cumulative positive effect onplant growth, presumably because photosynthate can be supplied to sinktissues earlier than in non-transformed controls. Enhanced growth isorderly, with no differences in organ morphology and with no evidencefor neo- or hyperplasia.

The foregoing description of the invention is exemplary for purposes ofillustration and explanation. It should be understood that variousmodifications can be made without departing from the spirit and scope ofthe invention. Accordingly, the following claims are intended to beinterpreted to embrace all such modifications.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 1                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Arabidopsis thaliana                                           - - <400> SEQUENCE: 1                                                         - - Tyr Phe Lys Asp Leu Gly Gly Met Pro                                       1               5                                                          __________________________________________________________________________

What is claimed is:
 1. A method of producing a genetically modifiedplant characterized as having increased growth and yield as compared tothe corresponding wild-type plant, said method comprising:contacting aplant cell with a nucleic acid sequence comprising a functionalcycB1a;At regulatory sequence, wherein said regulatory sequence isoperably associated with a nucleic acid encoding a cyclin protein, toobtain a transformed plant cell; producing plants from said transformedplant cell; and selecting a plant exhibiting said increased yield. 2.The method of claim 1, wherein the genetically modified plant exhibitsincreased root growth.
 3. The method of claim 1, wherein the geneticallymodified plant exhibits increased shoot growth.
 4. The method of claim1, wherein the cyclin is cyc1aAt.
 5. The method of claim 1, wherein theregulatory sequence comprises a cycB1a;At promoter.
 6. The method ofclaim 1, wherein the regulatory sequence comprises or hybridizes withthe sequence set forth in SEQ ID NO:1.
 7. The method of claim 6, whereinthe regulatory sequence comprises the sequence set forth in SEQ ID NO:1from about nucleotide -1 to about nucleotide -1148.
 8. The method ofclaim 6, wherein the regulatory sequence comprises the sequence setforth in SEQ ID NO:1 from about nucleotide -1 to about nucleotide -286.9. The method of claim 6, wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide -1 to aboutnucleotide -205.
 10. The method of claim 1, wherein the contacting is byphysical means.
 11. The method of claim 1, wherein the contacting is bychemical means.
 12. The method of claim 1, wherein the plant cell isselected from the group consisting of protoplasts, gamete producingcells, and cells which regenerate into whole plants.
 13. The method ofclaim 1, wherein said nucleic acid is contained in a T-DNA derivedvector.
 14. A plant produced by the method of claim
 1. 15. Plant tissuederived from a plant produced by the method of claim
 1. 16. A seedderived from a plant produced by the method of claim
 1. 17. Atransformed plant cell comprising a chimeric nucleic acid sequencecomprising:a cycB1a;At regulatory sequence operably linked to aheterologous nucleic acid sequence.
 18. The transformed plant cell ofclaim 17, wherein the regulatory sequence comprises or hybridizes withthe sequence set forth in SEQ ID NO:1.
 19. The transformed plant cell ofclaim 18, wherein the regulatory sequence comprises the sequence setforth in SEQ ID NO:1 from about nucleotide -1 to about nucleotide -1148.20. The transformed plant cell of claim 18, wherein the regulatorysequence comprises the sequence set forth in SEQ ID NO:1 from aboutnucleotide -1 to about nucleotide -286.
 21. The transformed plant cellof claim 18, wherein the regulatory sequence comprises the sequence setforth in SEQ ID NO:1 from about nucleotide -1 to about nucleotide -205.22. A transgenic plant comprising a heterologons nucleic acid sequenceregulated by a regulatory sequence of cycB1a;At wherein the regulatorysequence is operably linked to the heterologous nucleic acid sequence.23. The transgenic plant of claim 22, wherein the regulatory sequencehas, hybridizes with nucleic acid having, the sequence set forth in SEQID NO:1.
 24. The transgenic plant of claim 23, wherein the regulatorysequence comprises the sequence set forth in SEQ ID NO:1 from aboutnucleotide -1 to about nucleotide -1148.
 25. The transgenic plant ofclaim 23, wherein the regulatory sequence comprises the sequence setforth in SEQ ID NO:1 from about nucleotide -1 to about nucleotide -286.26. The transgenic plant of claim 23, wherein the regulatory sequencecomprises the sequence set forth in SEQ ID NO:1 from about nucleotide -1to about nucleotide -205.
 27. An isolated nucleic acid sequencecomprising a functional cycB1a;At regulatory sequence.
 28. The sequenceof claim 27, wherein the regulatory sequence comprises the sequence setforth in SEQ ID NO:1.
 29. The sequence of claim 28, wherein theregulatory sequence comprises the sequence set forth in SEQ ID NO:1 fromabout nucleotide -1 to about nucleotide -1148.
 30. The sequence of claim28, wherein the regulatory sequence comprises the sequence set forth inSEQ ID NO:1 from about nucleotide -1 to about nucleotide -351.
 31. Thesequence of claim 28, wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide -1 to aboutnucleotide -286.
 32. The sequence of claim 28, wherein the regulatorysequence comprises the sequence set forth in SEQ ID NO:1 from aboutnucleotide -1 to about nucleotide -205.
 33. The sequence of claim 28,wherein the regulatory sequence comprises the sequence set forth in SEQID NO:1 from about nucleotide -1 to about nucleotide -120.
 34. Thesequence of claim 28, wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide -1 to aboutnucleotide -60.
 35. A nucleic acid sequence comprising a functionalcycB1a;At regulatory sequence, operably linked to a nucleic acidsequence encoding a cyclin protein.
 36. A vector containing the nucleicacid sequence of claim 27 or
 35. 37. The nucleic acid sequence of claim35, wherein said regulatory sequence comprises the sequence set forth inSEQ ID NO:1.
 38. The nucleic acid sequence of claim 37, wherein theregulatory sequence comprises the sequence set forth in SEQ ID NO:1 fromabout nucleotide -1 to about nucleotide -1148.
 39. The nucleic acidsequence of claim 37, wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide -1 to aboutnucleotide -351.
 40. The nucleic acid sequence of claim 37, wherein theregulatory sequence comprises the sequence set forth in SEQ ID NO:1 fromabout nucleotide -1 to about nucleotide -286.
 41. The nucleic acidsequence of claim 37, wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide -1 to aboutnucleotide -205.
 42. The nucleic acid sequence of claim 37, wherein theregulatory sequence comprises the sequence set forth in SEQ ID NO:1 fromabout nucleotide -1 to about nucleotide -120.
 43. The nucleic acidsequence of claim 37, wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide -1 to aboutnucleotide -60.